IntroductionRice (Oryza sativa) is one of the world's most important cereal crops, providing staple food for nearly one half of the population. In many developing countries, rice is
the main source of food security and is intimately associated with local lifestyles and culture. With the rapid increase of global population, much greater rice production is
demanded, leading to wide application of transgenic biotechnology to rice for genetic
improvement1. Although no GM rice has been officially approved yet for extensive
commercial cultivation in the world, genes conferring traits, such as high amounts of beta-carotene, high protein content, disease and insect resistance, herbicide resistance, and salt tolerance, have been successfully transferred into different rice varieties through transgenic techniques2, 3. Some of these GM rice breeding lines or varieties
have been released into the environment for testing. It is apparent that as an important world cereal crop, transgenic rice varieties will inevitably be released into environments for commercial production in the near future.

Undoubtedly, biotechnology and GM crops will provide new opportunities for global food security and development in life sciences. However, the uses of GM crops have also aroused tremendous concerns about their biosafety world wide. The potential ecological risks associated with transgene escape through gene flow are the foremost among these concerns4.

When alien transgenes escape to and express in weedy or wild relatives of GM rice, transgenes may persist and disseminate within the weedy or wild populations through sexual reproduction and/or vegetative propagation. Transgenes that are responsible for resistance to biotic and abiotic stresses (such as disease and insect resistance, drought and salt tolerance, and herbicide resistance) can significantly enhance the ecological fitness of weedy and wild populations. The escape of these transgenes may cause ecological problems, for instance, by producing aggressive weeds, the spread of which might result in unpredictable consequences to local ecosystems. On the other hand, when transgenes escape to and persist in
wild rice populations, the rapid dissemination of transgenic hybrid individuals (or progeny) might change the original wild rice populations. In some cases, the aggressive spreading of hybrid swarms with better ecological fitness could even lead to the extinction of endangered
wild species populations locally5. Therefore, knowledge of the likelihood of gene flow from rice to its weedy and wild relatives will help to predict the magnitude of the
potential ecological consequences caused by transgene escape. This knowledge will also facilitate the effective management and safe use of the transgenic crops.

Cultivated rice and its weedy and wild relativesThe first step in the assessment of gene flow and its consequences is to determine which weedy and wild species can hybridize with the crop to produce fertile offspring. Cultivated rice is included in the genus Oryza of the grass family (Poaceae). This genus includes
two cultivated species (Asian rice Oryza sativa, and African rice O. glaberrima) and more than 20 wild species with ten different genome types, i.e., AA, BB, CC,
BBCC, CCDD, EE, FF, GG, JJHH, and JJKK. The wild relatives of rice with different genome types usually have significant reproductive isolation, making them unlikely to hybridize under natural conditions. Therefore, the wild species of concern for transgene escape are only those containing the AA genome.

As close relatives of cultivated rice, some wild rice species such as O. rufipogon, O. nivara, O. longistaminata, and O. glumaepatula are commonly found or coexist in rice farming systems of many Asian, African, and American countries. The weedy rice (also referred to as red rice, Oryza spontanea) is frequently observed in rice fields as an accompanying weed, particularly in the rice fields with direct-seeding cultivation practices. These AA-genome weedy and wild relatives are highly compatible sexually with cultivated rice. Their interspecific F1 hybrids could form complete chromosome pairing in meiosis and have relatively high pollen and seed fertility to produce viable offspring. Thus, studying gene flow from rice to its weedy and wild relatives becomes an important component for the potential ecological risk assessment of GM rice, because gene flow is the primary step from which potential ecological consequences of transgene escape may follow.

Gene flow from cultivated rice to its weedy and
wild relativesIn order to estimate the pollen-mediated gene flow from cultivated rice to its weedy and wild relatives, experiments were conducted at two sites in Kyongsan of South Korea and Chaling of Hunan Province, China, respectively, under special field conditions mimicking the natural
occurrence of weedy and wild relatives in Asia. Two types of experimental designs were established by constructing different populations to examine gene flow from cultivated rice
to weedy and wild rice species.

Gene flow from transgenic rice to weedy rice was measured using a transgenic rice variety (Nam29/TR18, as a pollen donor) with herbicide resistance (bar) and 13 accessions of weedy rice collected from Asia and America. The experimental plot was designed as complete random blocks where Nam29/TR18 was planted and mixed with one of the 13 weedy rice accessions in each block, respectively (Fig. 1).
Each block consisted of eight weedy rice plants. For identification of hybrids between Nam29/TR18 and weedy rice, seedlings generated from different weedy rice plants were sprayed with herbicide Basta at the 34-leaf stage. The surviving seedlings with resistance to herbicide Basta were considered hybrids and were subject to PCR detection of the herbicide resistance bar gene to confirm their hybridity. Gene flow frequencies were estimated by calculating the number of hybrids against the total number of seedlings germinated. The average frequencies of weedy rice seedlings with herbicide resistance were very low and varied among different blocks, but
with no significant differences among the replications. The experimental results indicated that the detectable rate of herbicide resistance gene flow from the transgenic rice to weedy rice plants varied between 0.011~0.046%.

Gene flow from cultivated rice to perennial common wild rice was measured using the Minghui-63 rice variety, and wild rice O. rufipogon (as pollen recipient)
was planted in different models to allow outcrossing to occur naturally. Co-dominant simple sequence repeats (SSRs) were used as molecular markers for accurate identification of hybrids between cultivated rice and O. rufipogon. The selected SSR primer pair amplified polymorphic alleles from the two species, which were easily distinguishable with electrophoresis in agarose gels. O. rufipogon presented a consistent fast-migrating allele (F) and Minghui-63 a slow-migrating allele (S) in the gels. The hybrids between the two species displayed stable heterozygous (FS) alleles. Leaf samples of germinated seeds from O. rufipogon populations were collected from individual seedlings
for SSR examination. Gene flow frequencies were estimated by calculating the number of seedlings with the FS heterozygote SSR pattern against the total number of seedlings examined. As a result, the frequencies of detected interspecific hybrids varied from 1.21~2.94% in different planting models. Gene flow frequency fromcultivated rice to O. rufipogon was therefore expectedly high, up to ca. 3%, although humidity and wind strength and direction significantly affected the rate of gene flow.

Potential consequences of transgene escape from GM rice to its weedy and wild relativesWith current concerns over weed problems caused by wild rice, and particularly the weedy rice in rice farming ecosystems, one of the major fears is whether the transgenes in GM rice varieties will escape to their wild and weedy relatives through gene flow, and enhance the fitness of the wild relatives. This could increase the weediness of
wild and weedy rice that invade rice fields, causing serious weed problems. Our experimental data clearly indicate the likelihood of gene flow from cultivated rice to its wild and weedy species, although with different frequencies. The gene flow frequency from cultivated Minghui-63 to wild O. rufipogon in different planting models varied between 1.1~2.94%. These frequencies are significantly high in terms of transgene escape if the cultivated GM
rice varieties are grown in the vicinity of wild rice species. Therefore, for the purpose of preventing or minimizing transgene escape to wild relatives, it is recommended that isolation zones with a sufficient space or with trap plants between GM rice and O. rufipogon should be established, until more effective methods are available. Effective isolation from GM rice will benefit the genetic integrity of in situ conserved wild rice populations.

The detected gene flow frequencies from GM rice line Nam29/TR18 to various weedy rice accessions were very low, ranging from 0.011~0.046% in one generation, when a weedy rice strain occurred simultaneously in a rice field. However, the gene flow frequency from cultivated to weedy rice in large populations might be more significant than the data observed in this experiment. Actually, rice cultivars cross easily with their related weedy forms
(red rice) found in direct-seeded paddy fields and produce viable and fertile hybrids with a reasonable rate. In addition, when weedy rice consistently occurs simultaneously with a cultivated rice variety in the same field, the number of hybrids resulting from gene flow could accumulate and increase through generations. If GM rice varieties are released to environments where weedy rice occurs abundantly, the transferred alien genes could spread and accumulate in weedy populations. This may pose a severe problem for weedy rice control and management in
rice production. Therefore, release of transgenic rice with genes that can significantly increase weediness and can resist weed control measures is not recommended in regions where weedy rice is already a serious weed problem.